Dietary fibre supplementation enhances radiotherapy tumour control and alleviates intestinal radiation toxicity

Background Non-toxic approaches to enhance radiotherapy outcomes are beneficial, particularly in ageing populations. Based on preclinical findings showing that high-fibre diets sensitised bladder tumours to irradiation by modifying the gut microbiota, along with clinical evidence of prebiotics enhancing anti-cancer immunity, we hypothesised that dietary fibre and its gut microbiota modification can radiosensitise tumours via secretion of metabolites and/or immunomodulation. We investigated the efficacy of high-fibre diets combined with irradiation in immunoproficient C57BL/6 mice bearing bladder cancer flank allografts. Result Psyllium plus inulin significantly decreased tumour size and delayed tumour growth following irradiation compared to 0.2% cellulose and raised intratumoural CD8+ cells. Post-irradiation, tumour control positively correlated with Lachnospiraceae family abundance. Psyllium plus resistant starch radiosensitised the tumours, positively correlating with Bacteroides genus abundance and increased caecal isoferulic acid levels, associated with a favourable response in terms of tumour control. Psyllium plus inulin mitigated the acute radiation injury caused by 14 Gy. Psyllium plus inulin increased caecal acetate, butyrate and propionate levels, and psyllium alone and psyllium plus resistant starch increased acetate levels. Human gut microbiota profiles at the phylum level were generally more like mouse 0.2% cellulose profiles than high fibre profiles. Conclusion These supplements may be useful in combination with radiotherapy in patients with pelvic malignancy. Video Abstract Supplementary Information The online version contains supplementary material available at 10.1186/s40168-024-01804-1.

The top four metabolites which had the lowest q-values in ANOVA test, followed by post-hoc analysis using Fisher's LSD and p-value adjustment using the Benjamin-Hochberg method, in each dietary group.Data is mean ± SEM.Supplementary Fig. 13 Unweighted UniFrac distance of faecal microbiota between cages in the radiosensitisation experiment.PERMANOVA test using the pseudo-F method was used to test for statistically significant inter-cage differences of the unweighted UniFrac distances of faecal microbiotas (n=5/cage) that were collected when tumours reached 700 mm 3 based on cages.For each comparisons, the first cage (dashed box) of each dietary groups, including (a) 0.2% cellulose, (b) psyllium, (c) psyllium plus resistant starch, or (d) psyllium plus inulin, was used as the control to measure its distance to the gut microbiota of the other eleven cages.For the control, only ten values were generated from the comparison of five mice with each other within the same cage.Twenty-five values were generated from the comparison of five mice from any two cages.Data are presented as mean±SEM.14 Weighted UniFrac distance of faecal microbiota between cages in the radiosensitisation experiment.PERMANOVA test using the pseudo-F method was used to test for statistically significant inter-cage differences of the weighted UniFrac distances of faecal microbiotas (n=5/cage) that were collected when tumours reached 700 mm 3 based on cages.For each comparisons, the first cage (dashed box) of each dietary groups, including (a) 0.2% cellulose, (b) psyllium, (c) psyllium plus resistant starch, or (d) psyllium plus inulin, was used as the control to measure its distance to the gut microbiota of the other eleven cages.For the control, only ten values were generated from the comparison of five mice with each other within the same cage.Twenty-five values were generated from the comparison of five mice from any two cages.Data are presented as mean±SEM.T cell function on tumours in responders and non-responders in psyllium plus inulin groups (n=3/group).The cell scores between responders and non-responders were compared by two-tailed t-test.The differential immunerelated gene expression analysis was followed the Benjamin-Hochberg method to correct p-values for controlling the false discovery rate (FDR).(c) Levels of plasma Th1 cytokines, GM-CSF and IL-2, stratified by IR.Correlations of these cytokines versus tumour growth rates in psyllium plus inulin group were assessed using the Pearson's correlation method.Data are presented as mean ± SEM.

a
Differences in bacterial components in the normal chow, 0.2% cellulose and psyllium groups.(a) Linear discriminant analysis (LDA) scores computed for differentially abundant taxa in the microbiomes of normal chow (grey), 0.2% cellulose (orange) and psyllium (red).Length indicates the effect size associated with a taxon.(b) Relative abundances of Bacteroides genus and Bacteroides caecimuris of different dietary groups compared with psyllium by ANOVA with Bonferroni's multiple comparison test. in bacterial components in the psyllium, psyllium plus RS and psyllium plus inulin groups.(a) Linear discriminant analysis (LDA) scores computed for differentially abundant taxa in the microbiomes of psyllium (red), psyllium plus RS (blue) and psyllium plus inulin (green).Length indicates the effect size associated with a taxon.(b) Relative abundances of Parasutterella genus and Faecalibaculum genus of different dietary groups compared with psyllium plus RS by ANOVA with Bonferroni's multiple comparison test.(c) Alpha diversity using Shannon's diversity index (a measure of community richness), Faith's phylogenetic diversity (a measure of community richness that incorporates phylogenetic difference between species) and Pielou's evenness (a measure of community evenness) of faecal microbiotas.One-way ANOVA with Bonferroni's multiple comparison test was used to compare the means of different dietary groups.Local tumour and systemic immune responses in all dietary groups.(a) Overall immune cell profiling evaluated on a NanoString platform.(b) Percentages of helper T and cytotoxic T cells measured by flow cytometry analysis.One-way ANOVA with Bonferroni's multiple comparison test was used to compare the means of different dietary groups.Data are presented as mean ± SEM.Supplementary Fig. 4 Discovery metabolomics analysis of caecal contents in all dietary groups.(a) Principal component analysis for caecal metabolites of different dietary groups.The left panel included the normal chow group, while the right panel removed it to obtain a better resolution for 0.2% cellulose and all psyllium-containing diets groups.(b) Survival analysis of tumour-bearing mice without and with IR in different dietary groups.(a) Overall and (b) individual Kaplan-Meier survival curve of mice with UPPL1591 allografts showing plots of time to quadruple in tumour volume.Phylogenetic composition of faecal microbiota when tumours reached 700 mm 3 .The sample sizes were n=5 in non-IR cohort and n=10 in IR cohort of each dietary group. in bacterial components in responders and non-responders in the psyllium plus inulin group.(a) Linear discriminant analysis (LDA) scores computed for differentially abundant taxa in the microbiomes of responders and non-responders.The alpha value was 0.05 for Kruskal-Wallis test and length of bar indicates the effect size associated with a taxon.(b) The relative abundances of Lachnospiraceae family and Muribaculaceae family between responders and non-responders were compared using two-tailed t-test.(c, d and e) Correlation between the Lachnospiraceae family, Muribaculaceae family and Bifidobacterium animalis versus the tumour growth in non-IR and IR cohorts of psyllium plus inulin.Tumour curve slopes were calculated by linear regression to represent tumour growth rates.The associations were assessed using the Pearson's correlation method.10 Principal coordinate analysis using Jaccard distance of faecal microbiota in the IR cohorts of (a) psyllium plus inulin or (b) psyllium plus RS.Faecal samples were collected when tumours reached 700 mm 3 (n=10/IR cohort in each dietary group).ADONIS test was used to assess the statistical significance of differences between the gut microbiota composition of responders and non-responders to irradiation within each dietary group.
between the gut microbiota versus the tumour growth in non-IR and IR cohorts of psyllium plus RS.Tumour curve slopes were calculated by linear regression to represent tumour growth rates.Associations between the tumour growth rates and the following variables: (a) Bacteroides genus, (B) Peptostreptococcaceae family, (c) Muribulaceae family, (d) P. burkholderiales or (e) Faecalibaculum genus were determined using the Pearson's correlation method.Supplementary Fig.12Beta diversity of faecal microbiota in the radiosensitisation experiment.Principal coordinate analysis using (a) unweighted (R 2 =0.5498,Pr(>F)=0.11)and (b) weighted UniFrac (R 2 =0.7975,Pr(>F)=0.11) of faecal microbiotas (n=5/non-IR cohort, n=5/IR cohort in each dietary group) that were collected when tumours reached 700 mm 3 based on cages (sphere, diamond and star denote three different cages) and dietary groups (orange-0.2%cellulose, red-psyllium, blue-psyllium+RS and green-psyllium+Inulin).ADONIS test was used to confirm the existence of significant differences among different dietary groups in terms of gut microbiota composition.
tumour cytotoxic T cells in the IR cohorts of all dietary groups.(a) IHC staining to assess the numbers of cytotoxic T cells in the irradiated tumours (n=10/group).(b) NanoString analysis of CD8 + cells and their ratio over T cells to assess the populations of cytotoxic T cells in the irradiated tumours (n=6/group).The expression of each immune cell's marker genes was normalised by a reference gene set and cell scores were calculated as the log2(average of normalised gene expression).One-way ANOVA with Bonferroni's multiple comparison test was used to compare the means among different dietary groups.Data are presented as mean ± SEM. tumour immune responses in the IR cohorts of all dietary groups and.(a) NanoString analysis of neutrophils and NK cells in the irradiated tumours (n=10/group).One-way ANOVA with Bonferroni's multiple comparison test was used to compare the means among different dietary groups.(b) Differential immunerelated gene expression between psyllium plus RS and psyllium plus inulin groups (n=6/group).The result was partitioned by psyllium plus RS.The Benjamin-Hochberg method to correct p-values for controlling the false discovery rate (FDR).A negative log2(fold change) indicates the gene overexpressed in psyllium plus RS group and a positive value indicates the gene overexpressed in psyllium plus inulin group.tumour and systemic immunity in psyllium plus inulin stratified by tumour response and IR.(a) NanoString analysis of exhausted CD8 cells and (b) gene sets of pathways of cytokines and receptors and 18 Correlations between the Clostridia and Lachnospirales orders and the populations of splenic (a) leukocytes, (b) macrophages, and (c) natural killer cells in the IR cohorts of psyllium plus inulin group.The associations were assessed using the Pearson's correlation method. a 20 Correlations between the caecal (a) threitol, (b) asparaginyl-hydroxyproline and (c) butyrate levels versus the tumour growth rate in IR cohort with or without non-IR cohort in the psyllium plus inulin group.The associations were assessed using the Pearson's correlation method.composition of faecal microbiota before and after irradiation in the acute toxicity experiment.Faecal samples were collected (a) pre-IR and (b) 3.75 days post-IR (n=3/non-IR cohort, n=6/IR cohort in each IR doses and dietary group).The samples are presented in the same order in the top and bottom panels.diversity of the gut microbiota and the metabolites profile in non-tumour-bearing mice after 3-week modified diet and 3.75 days after SARRP IR.(a) Principal coordinate analysis of faecal microbiota using Bray-Curtis dissimilarity.ADONIS test was used to confirm the existence of significant group differences in terms of gut microbiota composition.(b) Distances of gut microbiota in irradiated mice compared to non-IR controls in each dietary groups.PERMANOVA with the pseudo-F statistic was used to evaluate the statistical significance of differences in Bray-Curtis dissimilarity of gut microbiota among different IR doses.(c) Principal component analysis of metabolites profile in all and each dietary groups.ADONIS test was used to confirm the existence of significant group differences in terms of metabolite profiles.(d) Distances of metabolites profile in irradiated mice compared to non-IR controls in each dietary groups.The intensity of colour reflects the IR dose.ADONIS test was used to assess the presence of significant group differences in the Euclidean distance matrices of metabolite profiles among various radiation (IR) doses.26 Relative body weight of non-IR and IR cohorts of each dietary groups for the mice that did not receive IR or following IR.Body weight curves of (a) all and (b) single dietary groups were shown.The x-axis was the days after starting the modified diets.Data are presented as mean ± SEM.
27 Actual body weight of non-IR and IR cohorts of each dietary groups for the mice that did not receive IR or following IR.Body weight curves of (a) all and (b) single dietary groups were shown.The x-axis was the days after starting the modified diets.Data are presented as mean ± SEM.

Supplementary Fig. 5 Body weight changes of non-IR and IR cohorts of each dietary groups for the mice that did not receive IR or following IR. Body
weight curves of (a) all and (b) single dietary groups were shown.Slopes of body weight curves were calculated by linear regression and compared by ANOVA test and Bonferroni's multiple comparison test.Data are presented as mean ± SEM.

Supplementary Fig. Psyllium plus RS radiosensitised UPPL1591 bladder cancer cell allografts. Treatment
at day 26 post IR.Data shown as mean and standard error.Slopes of tumour curves were calculated by linear regression to represent tumour growth rates and compared by two-way ANOVA test and Bonferroni's multiple comparison test.Data are presented as mean ± SEM.

Supplementary Table 5 Rodent diets without corn starch used in the study with varying levels of cellulose, psyllium, psyllium plus resistant starch, or inulin per 4000 kcal.
resistant starch is based on the CoA of Hi-Maize Corn Starch, which has 62.8% TDF-dry basis and 11.7% moisture.